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UNIVERSITY OF CALIFORNIA 

COLLEGE OF AGRICULTURE 

AGRICULTURAL EXPERIMENT STATION 

BERKELEY, CALIFORNIA 

CIRCULAR 310 

November, 1927 

THE OPERATION OF THE BACTERIOLOGICAL 
LABORATORY FOR DAIRY PLANTS 

C. S. MUDGEi 




A typical bacteriological laboratory for a milk plant. 



INTRODUCTION 

Food stuffs generally are said to be perishable. By this we mean 
that sooner or later the material is rendered unfit or perhaps undesir- 
able for human consumption. It was Pasteur who found that the 
1 perishability ' of foods was due, not to a chemical change, but to the 
action of minute living bodies known as bacteria. Koch later devised 
a method whereby these bacteria might be counted, a method that is 
used today with certain changes. 



1 Associate Professor of Dairy Industry and Associate Bacteriologist in the 
Experiment Station. 



2 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

Milk is one of the foods that is considered highly perishable, since 
bacteria grow rapidly in it. The same constituents that make milk 
"the perfect food" for man also furnish the bacteria with an 
abundant supply of the most easily assimilated food. Roughly the 
number of bacteria in milk is a- measure of its quality and indicates 
the methods used in its production, so bacteriologists early began to 
study milk from the standpoint of the bacteria present. Here and 
there various workers began to tell of the numbers and kinds of 
bacteria they found. Unfortunately each man in his own laboratory 
employed methods which were largely his own. He used the solid- 
gelatin medium that Koch had told about, but the food material 
might be any soluble meat or vegetable extract that suited his fancy. 
With such a variety of methods the results obtained by different 
bacteriologists were not comparable. 

In 1873 a group of public-health officials and other prominent 
sanitarians formed the American Public Health Association to facili- 
tate the enlightenment of the general public on matters of public 
health and to act as a mentor for workers in this field. Acting in 
this capacity a committee in 1895 devised a standard method for the 
bacteriological examination of water. Every step in the procedure 
from sampling to the final report was carefully described. This 
proved so successful that those members of the association interested 
more particularly in milk, appointed a committee to formulate 
standard methods especially adapted to milk analysis. The findings 
of this committee have been published, and it is earnestly suggested 
that a cop}^ of the pamphlet 2 be obtained before an attempt is made 
to do bacteriological work on milk. 

The Standard Methods of Milk Analysis of the American Public 
Health Association, then, is the result of diligent work of these com- 
mittees. The methods are recognized as being, in theory at least, just 
what the name indicates — standard. It should be the aim of all those 
who undertake the enumeration of the bacteria in milk to adhere 
to these methods exactly. Some workers believe that the standard 
methods err. They are prone to attribute the least variation in the 
results to the method itself, forgetting the possible human factor 
which enters into all but the most exact methods. The methods used 
in enumerating bacteria in milk are not exact ; they are not intended 
to be. Newer knowledge of the science of bacteriology of course 
makes the method more and more dependable and from time to time 



2 American Public Health Association. Standard methods of milk analysis, 
5th ed., 40 pp. American Public Health Association, 370 Seventh ave., New York 
City. 1926. 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 3 

changes are made in the methods which in the opinion of the sponsors 
are for the best interests of all concerned. Much criticism, both just 
and unjust, has been directed to those responsible for the standard 
methods. It would be well, then, to study the reasons underlying the 
procedures which are suggested by these committees. Usually this 
criticism is directed toward the medium. Some laboratory worker, 
frequently unversed in the methods of research, collects the medium 
from several other laboratories and determines the colony count of a 
given milk sample upon these various media. His results show varia- 
tions and immediately he assumes that something is wrong. It is. 
The report of the committee appointed by the American Public Health 
Association upon the media in actual use by various laboratories shows 
alarming and absolutely unnecessary variations. Why, then, should 
absolute concord be expected when a choice of several peptones is 
made and when the amounts of all the ingredients used vary between 
wide limits? These criticisms of the medium are unjust — unjust 
until the real standard medium is used universally. Not only is this 
statement true, but there are other elements which also enter into 
these variations which in turn are ascribed to the medium. Two of 
these are the pipettes used and the dilution methods, which will be 
elaborated upon in their proper sequence. 

The purpose of this circular is to describe not only the methods 
used in the laboratory, but to evaluate the relative merits of the 
various methods which are used in the enumeration of bacteria in 
milk. It is written more especially for the plant man or the large 
dairyman, since the workers in the control laboratory of the city and 
state are usually previously trained to do this work. 



PLATE METHOD 

The plate method is essentially a device to trap bacteria in a thick 
jelly-like substance. In this substance the bacteria, not being able 
to move, develop with amazing rapidity in the place where they are 
trapped. This makes their progeny, all massed together in a single 
spot (the colony), visible to the naked eye, whereas the original "seed- 
ing" organism would pass unseen. Sterile apparatus, carefully 
measured dilutions, and cleanly methods in carrying out the operation 
are required. 

There are nine points or details of the method which are quite 
necessary to bear in mind all of the time when one is held responsible 
for the plating of milk for bacteria. 



4 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION 

These nine points, which will be discussed point by point, are: 
(1) the laboratory, (2) the equipment, (3) collection of samples, (4) 
transportation of samples to the laboratory, (5) the medium, (6) 
plating, (7) incubation, (8) counting, and (9) reporting. 

The Laboratory. — A laboratory may be either white-tiled through- 
out, equipped with polished metal autoclaves and stills, or it may be 
any room set apart from the rest of the plant for the sole purpose of 
controlling the quality of the incoming milk and of the efficiency of 



Incubator 



Scales 



'Store 





Sterilizer 



<4utoc/are 



O 



Fig. 1. — Plan of laboratory for a plant where both bacteriological and 
fat tests are to be made. 



the equipment used in the plant. The outlay for installation depends 
on the point of view of the management. The laboratory may be of 
the nature of a much advertised display, or an unassuming work room. 
The display idea is excellent, since it brings to the attention of the 
public the thought that the distributor is attempting to protect the 
quality of his product and the well-being of his patrons. Such a 
laboratory would, however, be expensive to install and maintain. 

Most plant laboratories are small, clean rooms, having perhaps a 
minimum of equipment, but nevertheless carrying out the funda- 
mental purpose of the laboratory control. It should be clean, light, 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 5 

accessible, and dry, should be large enough for the operator to work 
with ease, and should have a north light if microscopic work is to be 
undertaken. 

A room 12 feet by 14 feet seems about the right size. Anything 
smaller would handicap the analyst and the heat from autoclaves and 
sterilizers would be unpleasant. Figure 1 is a plan for a laboratory 
which is not far from ideal. The room has windows on two sides, 
one side being to the north. Since the work of the one in the labora- 
tory usually includes the Babcock test, this too is indicated. All of 
the apparatus that requires the use of steam or gives out much heat 
has been placed in one end of the room. Near the Babcock tester is 
the sink, with the stove and media-making apparatus beyond. The 
incubator is indicated as being below the table top in the corner. The 
plates are prepared on the table along the north wall, and are counted 
there. No space for an ice box is shown because it is assumed that 
media will be kept in the cold rooms of the plant. 

Electric flush receptacles should be generously installed at the 
time the laboratory is being finished. Table tops should be stained 
with aniline black. To do this, two solutions are made: 

Solution A — 

Aniline 120 grams 

HC1 (commercial) 180 grams 

Water 1000 cc. 

Solution B — 

Sodium dicliromate 120 grams 

Hydrochloric acid 100 grams 

Water 1000 cc. 

Solution A should be applied with a brush to the fresh smooth surface 
and allowed to dry overnight. The color will turn bright yellow. 
Solution B should then be spread on the wood, which will turn dark 
and be very streaky at first. After this second coat dries the surface 
should be rubbed with vaseline, motor oil, or paraffine. Vaseline 
seems to be preferable. 

In a dairy plant where there is plenty of steam, a connection 
should be made with the plant supply. Running water and electricity 
are essential. Gas is a desirable convenience, but can be replaced by 
electricity if not available. 

Equipment. — Below is given a list of the articles that are needed, 
together with the approximate cost. With this equipment properly 
set up an operator can do consistently good work if he understand the 
procedure. 



UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 



Article Number 

Autoclave (pressure cooker) 

Stove (2-3 burner) 

Double boiler 

Sterilizer (hot-air) 

Incubator, electric 

Scales 

Weights, 1 gram— 100 grams 

Funnels, 6" 2 

Funnels, 8" 2 

Funnels, 3" 6 

Bottles (6-oz. prescription ovals) 1 gross 

Bottles (2-oz. prescription ovals) 1 gross 

Graduates, 1000 cc 1 

Graduates, 500 cc 2 

Graduates, 100 cc 3 

Flasks, 1000 cc. Pyrex 24 

Flasks, 200 cc 36 

Petri dishes, 100 X 15 mm n* X 15 

Pipettes, 1 cc n 3 X 15 

Pipettes, 100 cc 2 

Pipettes, 10 cc 10 

Pipette box 2 

Counting lens 1 

Counting plate 1 

Counting hand tally 1 

Test tubes, 6" X %" 1 gross 

Thermometer, 10° to 100° C 2 

Thermometer, 0° to 250° C 1 

Microscope 1 

Breed pipette 1 

Microscopic slides 1 gross 



Cost per unit 
$35.00 

6.00 

2.00 
45.00 
80.00 

9.00 

2.00 

0.30 

0.40 

0.10 

4.50 

3.50 

2.00 

0.75 

0.35 

0.50 

0.30 

0.18 

0.18 

0.25 

0.20 

3.00 

3.00 

0.15 

5.00 

2.80 

2.20 

1.50 
125.00 

1.25 

1.35 



To this list should be addd as property constantly being replaced : 

Article Number Cost per unit 

Difco peptone lib. $6.00 

Filter paper 1 package 1.00 

Cotton absorbent, cotton batting, pinch cocks, rubber tubing, corks, etc., 

cork stoppers, wax pencils, pH indicator brom thymol blue, dropping 

bottles, stains. 



The equipment should include a steam sterilizer, which may be 
one of the autoclaves listed in the catalogues, or in a small laboratory 
it may be a pressure cooker such as the housewife has learned to use 
in the kitchen. If a pressure cooker is used, it should be fitted for 
steam by the shop mechanic to hasten the sterilization process. 



3 'n* in this case refers to the number of samples to be plated daily, 
samples daily, 6 X 15 = 90 plates needed to keep the work going. 



Six 



ClRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 7 

A dry sterilizer for glassware and an incubator are essential. For 
a hot-air sterilizer it is possible to utilize the common tin oven which 
can be purchased for a few dollars from the hardware stores. A 
wooden box can be used for an incubator if a thermostat is purchased, 
but it would be best to buy these pieces complete, since the equipment 
on the market is apt to be better and more efficient than makeshift 
appliances. However, if the plant has in its employ one who can 
construct such things it is possible to save some money. 

Such an array of chemical laboratory equipment might confuse 
the uninitiated, and the uses to which some of these are put would 
be difficult of description, so those to whom these articles are new 
would do well to visit some nearby health laboratory to familiarize 
themselves with their appearance and use. It is assumed that the 
reader has some knowledge of laboratory procedure. 

The petri dishes should be 100 mm. outside diameter with prefer- 
ably a 15 mm. side wall. Pyrex glass is expensive, but where it can 
be procured it would be worth the price paid because it lessens the 
breakage bill, which is high in most laboratories. The use of clay 
tops, in vogue ten years ago, is finding less support today. Not only 
do the films of media dry out more rapidly, but the ease of observing 
the dishes during incubation and during the mixing of the agar 
dilution is lost. 

In sterilization, the plates and pipettes should be placed in metal 
containers. A container 9" X 9" X 9" is a convenient size, since it 
will permit four piles of plates, 10 to a pile. Unless plates are steril- 
ized by the methods described there is danger of subsequent contam- 
ination through air. The plate should be kept in these containers 
until used. 

The straight-sided pipettes are best for bacteriological use since 
they are more easily placed in the containers. Right here is where 
the first chance for inaccuracy of plate counts is encountered. It has 
been found that the 1 cc. pipettes, as purchased on the market, fre- 
quently are inaccurate to the extent of 10 per cent or more. To guard 
against errors of this sort, each pipette should be filled with distilled 
water to the mark, and this water is weighed. It should weigh 
one gram ; if it is off as much as 10 milligrams in either direction, 
the pipette should be discarded. 

Another source of error is broken-tipped pipettes. The pipettes 
in general use today are made to deliver 1 cc, that is, there is but 
one mark upon the pipette, up to which the liquid to be measured 
is drawn. After drawing to this mark the liquid is allowed to flow 



8 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION 

out by gravity. It is obvious, then, that should the tip of the pipette 
be broken the pipette is useless where accurate amounts are to be 
delivered. The constant use to which these pipettes are put, the oper- 
ations of cleaning and sterilizing, and the shaking about while in the 
case, all tend to increase the liability of such breakage. The best 
way to overcome this source of inaccuracy is to use pipettes made to 
contain 1 cc, which have two marks, one well up on the pipette, the 
other near the tip. These pipettes are used in the same manner as 
the ones in general use with the exception that instead of allowing 
the liquid to flow out completely, it is allowed to run down to the 
lower mark. Any breakage of the tip with this pipette is of little 




Fig. 2. — Types of closures used for dilution bottles. The cotton plug is no 
longer much in favor. The wine cap (the center two) is used much in California 
and has many advocates. The screw-top bottle is used in this laboratory where 
it has proved of great help. 

consequence as far as the accuracy of the volume of the liquid de- 
livered is concerned. Such pipettes cost but little more than the usual 
types. 

Dilution bottles next claim attention. In figure 2 are shown 
various types of dilution bottles and of closures. The use of the 
cotton plug is to be discouraged, because the plug gets wet. Some 
laboratories use a rubber stopper and others the crown seal such as 
is found on soda bottles. This laboratory has tried many of the 
methods used to seal the dilution bottles and has adopted the screw 
finish bottle. The seals come in different sizes for the two sizes of 
bottles, so a yellow seal is used on the larger bottle, while a red one 
fits the smaller. Figure 2 also shows a type of closure very popular 
in laboratories in California. Wine caps such as were used on wine 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 9 

bottles in former days are admirable for closing dilution bottle; the 
chief objection is that the dilution water tends to leak out past the 
thumb which is used to hold the cap in place. Those workers favoring 
the wine cap claim, however, that this is no disadvantage. 

The dilution-bottle method offers another source of error unless 
reasonable care is used in measuring the water. The use of graduates 
for this work is to be deplored. A 100 cubic centimeter pipette is 
often used. In doing this, however, the 100 cc. level is marked on 
the lower end of the pipette. In use, the pipette is inverted and the 
water is drawn up easily through the larger opening. 

Automatic delivery pipettes in which one side of the apparatus 
fills while the other empties are much better, although their action is 
sometimes slow. 

In sterilizing the glassware dry heat should be used. The standard 
method calls for 175° C (347° F) for one hour, but such a time and 
temperature would leave one in doubt as to absolute sterility. A 
longer time at a little higher temperature, say two hours at 190° C, 
would be safer. This is the practice in the laboratory of the Dairy 
Industry Division. The dilution bottles should be sterilized in the 
autoclave as should the medium. 

Collection of Samples. — In the collection of samples too much care 
cannot be exercised, although too little attention is usually given to 
this operation. It is the connecting link between the milk to be exam- 
ined and the laboratory. 

In gathering samples a three-ounce screw-top bottle (see fig. 2) 
is best, although cotton-plugged tubes are still found in many 
laboratories. For taking the sample from the containers, a glass or 
aluminum tube is used. The tube is usually about 25-30 inches long 
with an inside diameter of a quarter inch. The relative merits of 
glass and metal tubes are more or less obvious. Glass tubes show 
whether they are clean or not, but they are prone to break. The 
question also arises whether a slender tube is capable of thoroughly 
stirring the contents of a can. Experiments conducted in this labora- 
tory indicate that representative samples can be taken by this means. 

The sample should be placed in ice unless it Is to be plated at once. 
Bottled milk should be shaken and sampled after very careful removal 
of the cap, during which operation the instrument used for opening 
should not pierce the lower surface. 

The Medium. — Before the samples reach the laboratory a supply 
of the medium which is to be used to grow the bacteria should be on 
hand. 



10 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION 

The standard medium has the following- composition: 

Agar 15 grams 

Peptone 5 grams 

Meat extract 3 grams 

Water 1000 cc. 

pH 6.8-7.0 

Distilled water should be used because it is more constant in its 
composition than tap water. In this should be dissolved the peptone, 
which is a soluble "predigested" meat product. The meat stuff from 
which it is prepared is composed of protein, which for the most part 
is insoluble. By digesting the meat with steam in an acid solution, 
the meat is broken down into its component parts: amino acids, pro- 
teoses, and peptones, hence the name. There are several peptones on 
the market, all of which are permitted in the preparation of the 
medium. It would seem that a better procedure would be to designate 
a single brand of peptone, rather than allow any one to be used. 
There are undoubtedly differences in brands and in their ability to 
support growth. Perhaps a way out of this difficulty would be to 
designate their composition or the stuff from which they are made. 

In the water is also dissolved a product called beef extract, which 
is the water extract of ground-up meat from which the water has been 
removed. Both of these ingredients, although composed of a varying 
mass of nitrogenous substances, nowadays are controlled in their 
manufacture so that a given brand is constant in its composition. 

Agar is next added to the water. Agar does not go into true 
solution but swells up and loses its shred-like structure, giving the 
medium a slightly cloudy appearance. 

After all of these have been added to the water, the hydrogen-ion 
concentration is determined. The determination of the hydrogen-ion 
concentration is not a difficult operation when once understood. It is 
somewhat different from the titrametric method (the phenolphthalein 
acid, test) in which a single indicator is used and acid or alkali 
added to bring the color of the solution to the 'neutral' point of the 
indicator. In the determination of the hydrogen-ion concentration 
several indicators may be used, all showing color changes but at 
diffent acidities. Without going into the basic theories of the reactions 
involved it can be well said that the idea is to determine the weight 
of hydrogen ions in a liter of water or solution. The thermometer, 
as is well known, is graduated in parts which are known as degrees. 
In a similar way the acid-alkali scale is graduated, so to speak, in 
degrees or in pH values. There are fourteen of these, from pII-0, 
which is a solution of normal acid (36.5 grams of concentrated HC1 



ClRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 11 

in 1000 cc. water), through pH 7 or the "neutral" point, to pH 14 
or normal alkali (40 grams sodium hydroxide in 1000 cc. water). 

It was mentioned above that for the colorimetric determination of 
hydrogen-ion concentration several indicators are used. Each of these 
indicators displays two colors — one for extreme acid and one for 
extreme alkali, say yellow for the acid and red for the alkali. There 
is some place in the pH scale where the transition from yellow to red 
takes place for each indicator. In this transition the intermediate 
blending of the yellow to the red colors make varying degrees of 
orange. Indicators have been selected which have these sensitive 
transition zones at various points of the pH scale from pH to pH 14. 
Figure 3 is given in an attempt to make the above statement clearer. 
In this figure the pH values are represented on the right. The vertical 
areas numbered 1 to 7 represent a hypothetical series of indicators. 
Each of these indicators is represented as having a yellow color for 
extreme acid (Y at top) and red for extreme alkali (R at bottom). 
These areas represent the sensitive zone where the indicator passes 
through changing orange colors from yellow to red. It will be seen 
that these zones overlap. In a solution of pH (fig. 3) indicator 1 
would be yellow. .All others would be yellow as well. If now we 
should add alkali, a drop at a time, to this solution, indicator 1 would 
slowly change to red as the pH became greater, or as the solution 
became more alkaline. But before the full red color of indicator 1 
develops indicator 2 would begin to change to red. This will be true 
throughout the pH scale, and demonstrates the general statement that 
every pH has some indicator which displays a telltale color by which 
that pH can be determined. 

With this explanation in mind, let us assume that we have a 
solution A which is pH-0.5. Indicator 1 will be yellow with a slight 
trace of red in it, a very yellowish orange. Every other indicator 
will be wholly yellow. Taking in order indicators B, C, D, etc., table 1 
gives the color each indicator will be at that pH. 

In actual practice we do not have all indicators of the yellow-red 
variety, nor do we use the whole range pH to pH 14. In biology, 
in general, and in bacteriology, in particular, the range is usually 
from pH 4.0 to pH 9.0 and we use about 4 indicators to cover the 
range. To narrow it down still more, the media-maker is concerned 
with but one indicator and its color changes. This is represented by 
indicator 8 in figure 3, or Brom thymol blue which is yellow at pli 6 
and blue at pH 7.5, with all shades of green between. To aid the 
worker unfamiliar with such things an artifice is employed which 
closely accords with absolute values known to more exact work. 



12 



UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION 

\o 



D — 



fellow 



Yellow 



ted 



Y 



Red 



Yellow 



■Meufral point 
Y 



Blue 



Y 



Y 



I 

a 

3 
A 
5 
6 

7 \ 

S 

9 

10 

II 

IE 

13 
14 



Fig. 3. — The theory of the use of several indicators. Each indicator changes 
from its full acid color to its full alkaline color at definite narrow pll ranges. 

TABLE 1 

Colors that Various Indicators Shown in Figure 3 Will Have at 

Points A-B, etc. 





pHof 
solution 


Indicator 


Point 


1 


2 


3 


4 


5 


6 


7 




will be 


B 


1.6 


Red- 


Yellow 


Yellow 


Yellow 


Yellow 


Yellow 


Yellow 


C 


4.7 


orange 
Red 


Red 


Yellow- 


Yellow 


Yellow 


Yellow 


Yellow 


D 


6.7 


Red 


Red 


orange 
Red 


Yellow- 


Yellow 


Yellow 


Yellow 


E 


8.7 


Red 


Red 


Red 


orange 
Red 


Yellow- 


Yellow 


Yellow 


F 
G 


10.7 
12.7 


Red 
Red 


Red 
Red 


Red 
Red 


Red 
Red 


orange 
Red 
Red 


Orange 
Orange 


Yellow 
Orange 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 



13 



A series of six tubes is placed in a test tube rack and to these tubes 
is added a weak solution of hydrochloric acid (1 drop of acid to 100 cc. 
of water) 5 cc. to a tube. Another set of six tubes is placed in front 
of these into which is placed 5 cc. of 0.5 per cent sodium hydroxide 
solution. To one tube of the acid is added 9 drops of the indicator 
Brom thymol blue, to the next 8 drops, then 7 drops, etc. It will be 
seen that there is a decreasing intensity of the yellow color as less 
and less of the indicator is used. Now to the series of alkali tubes 1 
drop of indicator is added to the first tube, two to the second and so 
on, which gives an increasing intensity of blue as more of the indicator 
is added. 

A B C D E r 



Acid 
Tubes 



I 

0^ 



I 



'a 






i 

<0 






\ 



Alkali 

Tubas 



! 



(VI 



! 



E. 






I 



a. 



I 



pH 6.2 6.4 6.7 6.9 7.1 7.J 

Fig. 4. — The drop-ratio method of determining pH values. 



When this is done there will be six pairs of tubes, each pair will 
have 10 drops of indicator between them (9 plus 1, or 8 plus 2, etc.). 
It so happens that on looking through any pair of tubes the shades 
of green will correspond to certain pH values. 

Figure 4 brings this out more clearly. The pair of tubes marked A 
(reading down) having between them 10 drops of indicator will have 
a color corresponding to pH 6.2 when they are observed one behind 
the other. To better perform this operation a comparator block is 
used which is shown in figure 5. The tubes are inserted and observed 
through the holes bored in the side of the block. Figure 6 shows the 



14 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

block ready for use. In this figure the six pairs of tubes described 
above are shown, one pair, however, is in the comparator block. 
Behind this pair is placed a tube of medium since it normally has a 
color of its own which must be taken into consideration in matching. 
In the block is also placed a tube containing 1 cc. of the medium, 4 cc. 
of water and 10 drops of indicator. Behind this there are frequently 
placed two tubes of water, although this is not necessary. 

If the color of the indicator in the medium does not correspond to 
the color of the tubes between pH 6.6 to pH 7.0 adjustment is neces- 
sary. To adjust the medium N/20 4 NaOH is added from a burette 







Fig. 5. — The comparator block. 

or pipette into the tube until the desired shade of green is obtained. 
Fifty times this amount of N NaOH is now added to a liter of the 
medium which will be found to be of the desired pH. 

The medium is now placed in flasks or bottles in 100 cc. quantities, 
and sterilized in the autoclave for 25 minutes at 15 pounds pressure. 

When time is at a premium dehydrated media can be used. This 
is a complete medium with the water extracted. All that is necessary 
for preparing a liter of standard agar is to weigh out the required 
amount of the dry powder, dissolve, and sterilize. The initial cost of 
this medium is higher than that prepared as given above. In a busj' 

* N/20 NaOH is made by dissolving 2 grams of sodium hydroxide in 1000 cc. 
of water. N NaOH is 20 times stronger. For such work as described above these 
solutions would not have to be corrected, although correction would be necessary 
if refined work were done. 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 



15 



plant laboratory where the operator is required to do work other than 
that of a strict bacteriological nature, this extra cost would not be a 
factor of importance. 

A method nearly as rapid is recommended by standard methods. 
A concentrated agar solution is made. This is melted when agar is 
needed, and a concentrated solution of the peptone and extract is 
added to it, For this, the agar for 1 liter is dissolved in 600 cc. of 
water while the nutrient (peptones, etc.) for the liter is dissolved in 
400 cc. of water. The agar, however, is made up in large quantities 
and stored in 600 cc. amounts. Whenever a liter is needed the nutrient 
solution is made up and added to the agar. 




Fig. 6. — The color standards and comparator block in use. 

Sometimes for special work in bacteriology, sugars and other 
fermentable substances are used for diagnosis, and aiding in the 
growth of the colonies. Lactose is of great importance, especially 
in studying milk bacteria. However, although there are some who 
believe that the medium would be greatly benefitted by such an 
addition, it is not standard practice. 

Plating.— On arrival at the laboratory of the samples to be tested 
the actual work of plating should be started at once, especially if 
the samples mentioned are to be the only lot received that day. 
Should there be several inspectors collecting samples, as is the case 
in large city laboratories, it is permissible to wait for all samples to 
be brought to the laboratory, provided they are kept at a temperature 
below 40° F. The samples must be completely covered with ice in a 
closed container to bring them down to that temperature. 



16 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

Having then sterilized all equipment, and with the samples to be 
plated at hand, the analyst is ready to begin. The table top should 
be thoroughly wiped with a damp cloth to remove the dust that may 
have collected there. The plates, dilution bottles, and samples should 
be carefully arranged along the table. First from the edge should be 
placed the petri dishes; next the sample to be plated, and lastly the 
dilution bottles. The petri dishes should then be marked with sample 
numbers, date, and dilution. This laboratory has used a method of 
marking the petri dishes which is worthy of mention. Instead of the 
long rows of ciphers it would be necessary to write down if a plate 
were marked " 100,000' ' or "1,000,000" the following shorter, more 
easily read system is used: 100 becomes H, 1,000 becomes T, 10,000 
becomes 0T, 100,000 becomes 00T, 1,000,000 becomes M. Also one 
will frequently read "100,000" as though it contained one cipher 
more or less. 

Assuming everything is now in readiness, the plating begins. The 
theory of the procedure is that, since it would be impractical to count 
the bacteria in a whole cubic centimeter of milk, a fraction of a cubic 
centimeter is used. The fraction, 1/1,000 or 1/1,000,000 of a cubic 
centimeter, is chosen according to the analyst's estimate of the prob- 
able germ content of the milk. To get these fractions of a cubic 
centimeter (cc), the milk is mixed with sterile water. One could, 
of course, in making a dilution of 1-1,000,000, take a single cubic 
centimeter and add it to 1,000 liters of water or 264 gallons and take 
a sample of that. Such methods would be absurd, so a system of 
progressive decimal dilutions are made. In so doing, taking 1 to 
1,000,000 dilution again as an example, 3 bottles are used, each con- 
taining 99 cc. of distilled water. One cc. of milk is added to the first 
99 cc. of water. The bacteria in this 1 cc. of milk are then floating 
about in 100 cc. of fluid (1 part milk and 99 parts water) which is 
called the "l-to-100 dilution." If this in turn is again diluted by 
adding 1 cc. of the l-to-100 dilution to a second 99 cc. of water the 
bacteria will be floating in a fluid composed of 1 part milk and 9999 
parts water and is the l-to-10,000 dilution. This can continue until 
several 99 cc. portions are taken. The jump from a dilution of 1-100 
to 1-10,000 is large, so it is the practice in laboratories to use a 9 cc. 
dilution between two 99 cc. dilutions. 

Table 2 is given to more fully explain the process of making the 
dilutions. The first 99 cc. dilution receives the milk from the sample. 

A little study of the above table should make the procedure clear 
to anyone. It is not necessary to make the first 9 cc. dilutions if 
dilutions of 1-100,000 are to be made nor the second 9 cc. dilution if 



ClBC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 



17 



1-1,000,000 are desired. It is wise unless one is certain of the prob- 
able number of bacteria in a sample of milk to make at least two 
dilutions. 

TABLE 2 
Outline of Method Used in Making Dilutions 



Step 


Source 


Dilution bottle 
used 


Resulting dilution 


Mark on 
Petri dish 


A 


Sample 


99 cc 


1-100 


H 


B 


1-100 dilution 


9 


1-1000 


T 


C 


1-100 dilution 


99 


1-10,000 


OT 


D 


1-10,000 dilution 


9 


1-100,000 


OOT 


E 


1-10,000 dilution 


99 


1-1,000,000 


M 



The standard methods require that the original sample, as well 
as the dilutions, should be shaken 25 times before subsequent portions 
are removed. The reason for this is evident when it is known that 
frequently the bacteria in milk are found as streptococci and strepto- 
bacilli, in which chains are formed containing a number of cells. If 
the dilution is made without shaking, the whole chain may be seeded 
into the agar and grow into a colony to be counted as one. On the 
other hand, if the dilution is shaken excessively the chains are entirely 
broken up, causing as many colonies to form as there are individuals 
in the chain. Since either extreme might be used, and since excessive 
shaking cannot be easily denned, a middle course is taken, consisting 
of shaking 25 times, each shake being an up-and-down motion of 
about one foot. Let it be repeated at this time that in the process 
of plating, sterile glassware, sterile dilution water, and sterile media 
should be used throughout. 

After the sample is shaken, the cap should be removed without 
piercing the paper, and one cubic centimeter of the milk removed. 
This is transferred to a dilution bottle which is shaken as directed. 
The pipette should not be blown out; the fluid should be allowed to 
flow out of its own accord. It should not be rinsed in the dilution. 
Dilutions are thus made until one is reached which, in the opinion of 
the analyst, is correct for that particular sample. One cubic centi- 
meter of the milk-and-water dilution is then transferred to a petri 
dish properly marked. 

At once, or within 20 minutes at the most, the agar is poured upon 
the milk dilution and the two mixed thoroughly with a gentle rotary 
motion. The agar is then allowed to solidify, after which the plates 



18 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

are placed in the incubator in an inverted position. This inverting 
of the plates is a precaution against 'spreaders.' If the plate is placed 
agar-side down, the drops of condensation water falling on the plate 
might cause contamination if it should strike a colony. By inverting 
the plates the water that does condense remains where it formed. 

Incubation. — In order to stimulate the growth of the bacteria 
seeded in the agar, the plates are incubated at 37° C. The time for 
incubation has also been specified by the American Public Health 
Association as 48 hours and should be adhered to closely. The plates 
in the incubator should be stacked not too closely, because close stack- 
ing leads to inaccuracies in the final count. It has also been suggested 
that a fan be placed in large incubator rooms to keep the air in 
motion. The purpose of this is not to create a draft, but simply to 
overcome stratification of air. The temperature of the air at various 
places about the incubator should be determined. The incubation of 
the plates is one of the most important steps. 

Counting Plates. — At the end of the incubation period, the plates 
should be removed to the laboratory and the colonies counted. 
Theoretically, each colony results from the growth of a single organ- 
ism. The number of colonies is then multiplied by the dilution, but, 
if two dilutions have been made the one is chosen that contains from 
30 to 300 colonies. This is done because it has been found from 
experience that plates with less than thirty colonies give results that 
are high, and when there are more than 300 colonies to a plate there 
is apt to be crowding. Products of growth from one colony diffuse 
through the medium and frequently prevent bacteria in other colonies 
from growing. This sometimes gives rise to the formation of "pin 
point" colonies. 

In counting plates a dark background is used such as is pictured, 
in figure 7. Some laboratory workers prefer a highly illuminated 
background but the author has found the dark background very satis- 
factory. The counting is done with a hand lens magnifying 2% 
diameters. 

Standard Methods of Milk Analysis discusses the common sources 
of error in plate counts as follows : 

"Agar plate 'counts' per cc. are to be regarded as estimates of 
numbers rather than as exact counts, since only a portion of a cubic 
centimeter is used in preparing the plates. As such they are (like all 
estimates) subject to certain well known and recognized errors whose 
size can be largely controlled by the care taken in the analysis. 
Among these errors are: (a) Failure of some of the bacteria to grow 
because the incubation temperature or the composition or reaction 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 



19 



of the medium is not suitable. (&) Inaccuracies in measurement of 
the quantities used, (c) Mistakes in counting, recording data, com- 
puting results, and the like, (d) Incomplete sterilization of con- 
tamination of the plates, dilution waters, etc. The possible errors 
caused by these things makes it highly important for all routine 
laboratories to follow carefully a standard procedure. 

''Recent investigations make it clear that these largely controllable 
errors are not so likely to cause misconceptions of the accuracy of the 
results as are the errors due to the fact that bacteria in milk usually 
cling together in groups of from two to many hundreds of individuals. 
These groups are only partially broken apart by the shaking given in 
preparing the dilutions, so that at best the counts from the agar plates 




Fig. 7. — The counting plate, the tally counter, and the magnifying glass 
used in counting colonies. 

represent the number of isolated individuals and groups of two or 
more bacteria that exist in the final dilution water. Thus the colony 
counts from the plates are always much smaller than the total number 
of bacteria present. This error would not be troublesome if the 
groups were of constant average size, but the best information avail- 
able shows that the groups in ordinary market milk commonly vary 
in size so that they contain an average of from 2 to 6 individual 
bacteria. Some samples contain groups of even smaller size than this, 
while others, such as those bearing long-chain streptococci, may show 
groups containing an average of 25 or even more individual bacteria. 
The irregularity of this error (whose size is not indicated in any way 
by the appearance of the plates) should be kept in mind in interpret- 
ing the results obtained. 

"Because of the fact that agar-plate counts only represent a 
fraction of the total number of bacteria present, they should not be 
reported as showing the 'number of bacteria per cc' Accurately 



20 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

speaking, the counts from agar plates give the estimated number of 
colonies that would have developed on standard agar per cc. of milk 
if an entire cubic centimeter of milk had been used for inoculation. 
Because this statement of fact is cumbersome, and also because a 
certain ratio exists in each case between the colony count and the total 
number of bacteria, it has become a common practice to speak of the 
plate counts as showing the number of bacteria per cc. This is very 
confusing now that microscopic methods of counting have been devel- 
oped which permit counts of the actual bacteria to be made. The 
most frequent ratio between agar-plate counts made by the official 
plating method and the total number of bacteria present has been 
found to be approximately 1 to 4. 

"It is therefore recommended that all agar plate counts obtained 
by the standard technique shall not be reported in the form ' 2,000,000 
bacteria per cc. ' but rather as follows : ' Official plate count, 2,000,000. ' 
The latter form of expression shall be considered an abbreviated 
method of saying: 'a count of 2,000,000 colonies per cc. as obtained 
by standard methods.' Moreover, analysts shall be careful to avoid 
giving a fictitious idea of the accuracy of the official plate count. 
There is ample justification for thinking it sufficiently accurate to 
justify drawing conclusions as to the general quality of a given 
sample of milk, and when a series of samples from the same source 
are examined the average result may permit much more specific con- 
clusions to be drawn with confidence." 



THE MICROSCOPIC COUNT 

The microscopic count, also known as the Breed or direct count, 
is the method sponsored by Dr. R. S. Breed, in which the bacteria 
themselves are seen directly by means of a microscope. The method 
was developed owing to a demand on the part of milk-plant operators 
for a more rapid method than the incubated-plate method. There 
have been many criticisms of the plate method. These criticisms 
usually are directed to the facts that all of the bacteria do not grow 
on the media used, that it takes 48 hours to obtain results, that the 
underlying principle of colony growth from a single organism is not 
always true, and that many of the operations are subject to relatively 
great personal errors on the part of the analyst. 

The plate method, regardless of the possible truth of the above 
criticisms, has been used for many years and around its findings has 
been developed a certain well-defined conception of what constitutes 
a good milk from the bacteriological point of view. Any method, 



ClRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 21 

whether it is a direct enumeration of the bacteria seen by the micro- 
scope or whether it involves such growth phenomena as reductase 
production, must obviously be correlated to the older concepts that 
the plate method has developed. 

Anyone can make a smear of milk on a glass slide and see the bac- 
teria after staining. The significance of the numbers of bacteria 
seen is of greater importance. They must be thought of in terms of 
the unit of measure, the cubic centimeter. 

This difficulty was obviated by controlling the possible variable 
factors that enter into the method. A definite quantity of milk 
(0.01 cc.) is spread over a definite area (1 sq. cm.), which gives a 
film approximately 0.1 mm. in thickness. This film, after staining, is 
examined with a microscope having a field of view of a definite size 
(0.205 mm.). The milk film observed through the microscope is 
1/300,000 of a cubic centimeter. From the number of bacteria seen in 
this amount of milk the number in the whole cubic centimeter can 
be easily calculated. 

Pipettes are now sold which are made to deliver 0.01 cc. of milk. 
Care should be exercised in obtaining these, only those being chosen 
which have a ground-off, cone-shaped tip. The drop should be dis- 
charged cleanly and should not run back on the side of the tip. 
These pipettes vary somewhat and it would be well to calibrate them 
by determining the weight of milk contained in the pipette. It should 
deliver 0.0103 gram. 

A single pipette is needed for the work. It need not be sterile 
but should be flushed out after each sample of milk by rinsing in 
warm clear water. The pipettes when not in use should be kept in 
a glass-cleaning solution or in sulphuric acid. 

The slides used may be the common 1" X 3" microscope slide. 
If these are used, a card can be purchased which serves as a guide 
in the spreading of the milk. Slides somewhat larger in size but with 
the surface ground except for little windows of the proper size, are 
also in use. This may be more easily visualized by referring to 
figure 8. Figure 8 a shows a microscope slide with the film of milk 
spread over 1 sq. cm. Figure 8 b shows a cross section along line 
A-B (fig. 8 a) with the milk film represented. The microscope 
objective is just above the film over that portion which is .shaded. 
This shaded portion is next shown (fig. 8 c) as a little pellet or button 
of milk 0.205 mm. in diameter and 0.1 mm. thick. This button is 
1/300,000 of a cubic centimeter. From the number of organisms seen 
in this button (5 in this case) the number per cubic centimeter can 
be calculated by simple multiplication. 



22 



UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 



The steps in the microscopic method are as follows : 

1. The milk is drawn into the pipette and the tip of the pipette is 

wiped with a clean cloth. 

2. The milk is blown onto a glass slide. 

3. It is spread over an area of a square centimeter by means of a 

sterile wire. 

4. It is dried, care being taken not to dry too quickly because such 

drying causes cracking. 

5. The slide is placed for one minute in a Coplin jar containing 

xylol to remove the fat from the film. Gasoline, or any other 
fat solvent, may be used, but the xylol is suggested in the 
standard methods. 

6. The slide is drained and dried. The film now has a frosty white 
appearance. 




Milk film 
O.lmm thick 



Objective 



O/oss s/ide 




Fig. 8. — Top: Slide with milk film spread over 1 square centimeter. Middle: 
Side view showing milk film magnified with objective. The shaded area is the 
portion of milk film seen at any one time. Bottom: Greatly magnified portion of 
film showing that portion of film seen at any one time. It is 1/300,000 of a cubic 
centimeter. 



CIRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 23 

7. The slide is then placed in a second Coplin jar containing 90 

per cent grain alcohol or denatured alcohol. 

8. It is next placed for at least one minute in a Coplin jar 

containing methylene-blue solution prepared as follows : 
saturated alcoholic solution of methylene blue, 30 cc., 5 0.01- 
per-cent solution of caustic potash, 100 cc. The film is 
purposely left in the bath for a long time to overstain. 

9. The film is decolorized by again placing the film in the alcohol. 

The alcohol draws the stain from the milk more rapidly than 
it is drawn from the bacteria, thus giving a contrasting 
picture. 
10. The film is dried and examined. 
Before the film can be examined with any assurance that the find- 
ings will be comparable to our previous understanding of what 
bacterial numbers in milk means, the microscope must be adjusted. 
For those to whom the microscope is new, a brief description of 
its parts may be of interest. In figure 10 is shown the parts of a 
laboratory microscope. To the stand is attached a mirror, a con- 
denser, a stage, and a body. The body has a nose piece with usually 
three objectives (lens near the object to be viewed) and a selection 
of eyepieces, only one of which is in use at one time. By varying 
the eyepieces and objectives one can approach the required size of 
the field, 0.205 mm. The final adjustment is made by pulling out 
the draw tube. The steps in the calibration of the microscope are 
simple to one who understands microscopy. To the layman, however, 
it would be difficult to explain, and explanation is, indeed, unneces- 
sary, since in the purchase of a microscope the buyer can request the 
desired calibration of the seller. A mechanical stage, which is an 
attachment permitting very slow and controlled movements of the 
slide upon the stage, is an absolute necessity to good work. These 
directions are given to aid in understanding the methods, but no one 
should attempt the Breed method without working at least a day with 
someone versed in the technique. 

The picture one sees when looking into the microscope is hard 
to describe. There may be rods and cocci, as well as objects appearing 
similar to these. One has to become accustomed to what is supposed 
to be seen. The cells must be counted in each of thirty fields, the 



5 There are many brands of stain upon the market today, and some of these 
are better than others. To protect the users of such products from spurious and 
cheap articles a commission headed by Dr. H. J. Conn examines every batch of 
stain produced by manufacturers. If the stain is found to pass the rigid inspec- 
tion of the committee a label bearing the signature of Dr. Conn is attached to 
the bottle. Do not use stains not bearing this label. 



24 



UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 



average taken, and this average multiplied by 300,000; or the total 
for thirty fields may be counted and this total multiplied by 10,000. 
The results in either case are the same. Although the regulation 
number of fields to be counted is 30, there are frequently occasions 
when averaging but 5 fields will give all the information necessary. 
Practice is necessary to successfully use the Breed method. 



E— Eyepiece 



Pinion Henri — 




[) S Stage 



SS — Substage 



Inclination J 



Mechanical Features of Microscope 

Fig. 9. — The parts of a microscope. 

The method will not permit too close separation of grades of milk. 
One could not, in other words, distinguish between a milk with 18,000 
bacteria per cc. and one with 22,000. But for grading milk into larger 
groups nothing is simpler, nor more rapid. For each locality the 
ranges of a passable milk would change slightly, and these limits 
should be established for each locality. 

In the beginning of this discussion on the Breed method it was 
considered that a parity should exist between it and the established 



ClEC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 25 

plate method upon which our laws and regulations have been based. 
Dr. Breed has had a number of workers study the method to obtain 
just this information. 6 As a result of this work, it is now thought 
that the direct count is, as a rule, four times greater than the plate 
count. This being accepted, it is necessary to divide by four the count 
obtained by the direct method. This will give a value close to that 
of the plate count. 

THE FROST LITTLE-PLATE METHOD 

The Frost method of enumeration of bacteria in milk is a com- 
bination of the plate and the direct methods. Milk and agar are 
mixed in equal portions and 1/20 of a cc. is spread over an area of 
4 sq. cm. This slide is placed in the incubator until the colonies 
develop. It usually takes about 8 hours. The microscopic colonies 
are then counted after the film is dried and stained as in the method 
used in the direct count. No new principle is put to use, and, since 
the method is not much in vogue except in special cases where large 
city plants hold pasteurized milk before delivery, it will not be de- 
scribed in detail. 

THE REDUCTASE TEST 

Many dyes when added to milk in dilute solutions will be decolor- 
ized. Litmus and methylene blue will do this. There seems to be a 
feeling that there is an indirect relation of the rate of the decoloriza- 
tion and the number of bacteria; the fewer the bacteria the longer 
the time necessary for decolorization. This is only generally true, 
since not all bacteria possess this property of decolorization. It is 
apparent that a milk might have a large number of bacteria present 
and still not be decolorized. However, the method is one that has 
found considerable support on the continent of Europe and even in 
this country the idea is growing. The argument usually put forward 
is that we have a medium in which the bacteria can grow best (the 
milk itself) instead of a highly artificial one such as the usual nutrient 
agar. Be that as it may, the classes into which milk is grouped has 
no great significance is California, where the milk is of a better 
quality generally than in many parts of the country. The following 
classes are recognized in Europe : 

Class 1. — Good milk, not decolorized in five and a half hours, 
containing, as a rule, less than one-half million bacteria per cubic 
centimeter. 



6 Robertson, A. H. 1921. See "Selected list for further reading.' ' 



26 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

Class 2. — Milk of fair average quality, decolorized in less than five 
and a half hours but not less than two hours, containing as a rule, 
one-half to four million bacteria per cubic centimeter. 

Class 3. — Bad milk, decolorized in less than two hours, but not 
less than twenty minutes, containing, as a rule, four to twenty million 
bacteria per cubic centimeter. 

Class 4. — Very bad milk, decolorized in twenty minutes or less, 
containing, as a rule, over twenty million bacteria per cubic centi- 
meter. 

However, we still have use for the method under certain conditions 
and the method is described. 

Methylene-Blue Solution. — A stock solution of methylene blue is 
made by dissolving 1.1 grams of the dry powder in 500 cc. of distilled 
water. One cc. of this stock solution is diluted with 39 cc. of distilled 
water, giving a concentration of 1 part of dye to 20,000 parts of 
water. One cc. of this added to 10 cc. of milk makes a final dilution 
of the dye in the milk of 1 to 200,000. The color is a robin's egg blue. 

Ten cc. of milk are placed in the test tubes and 1 cc. of the methy- 
lene-blue solution added. Sterile tubes are not a necessity but they 
must be clean. The contents are thoroughly mixed and placed in a 
water bath at 37.5° C. The tubes are observed at intervals, and notes 
are made on the time of decolorization. An exact agreement between 
the time taken to reduce the dye and the plate count is not to be 
expected. 

There are other methods of evaluating the bacteria content of a 
sample of milk. These will not be discussed, because they are little 
used. Those that have been explained are the ones in most general 
use. Each has advantages; each has faults. The plate method is 
slow but accurate if carefully done. The direct count is rapid but 
suffers by reason of the factor of 300,000 which has to be applied. 
The little-plate method (Frost's) has the inherent disadvantages of 
both the above. The reductase test has a splendid future by virtue 
of certain research that is being done in some of the laboratories of 
this country. 



ClRC. 310] BACTERIOLOGICAL LABORATORY FOR DAIRY PLANTS 27 



SELECTED LIST FOR FURTHER READING 

Plate Method 

EOBERTSON, A. H. 

1921. The relation between bacterial counts from milk as obtained by micro- 
scopic and plate methods. New York State (Geneva) Agr. Exp. Sta., 
Tech. Bui. 86:3-21. 
Ayres, S. H., and C. S. Mudge. 

1920. Milk powder agar for the determination of bacteria in milk. Jour. 

Bact., 5: 565-588. 
Brew, J. D., and W. D. Dotterrer. 

1917. The number of bacteria in milk. New York State (Geneva) Agr. Exp. 

Sta. Bui. 439:479-522. 
Brown, J. Howard. 

1921. Hydrogen ions, titration, and the buffer index of bacteriological media. 

Jour. Bact. 6:555-567. 
Barnett, G. D., and H. S. Chapman. 

1918. Colorimetric determination of reaction of bacteriologic medium and 

other fluids. Jour. Amer. Med. Assoc, 70:1062-1063. 
Breed, E. S., and W. D. Dotterrer. 

1916. The number of colonies allowable on satisfactory agar plates. New 
York State (Geneva) Agr. Exp. Sta., Tech. Bui. 53:3-11. 

Direct Count 
Breed, E. S., and J. D. Brew. 

1916. Counting bacteria by means of the microscope. New York State 

(Geneva) Agr. Exp. Sta., Tech. Bui. 49:3-31. 

1917. The control of bacteria in market milk by direct microscopic examina- 

tion. New York State (Geneva) Agr. Exp. Sta., Bui. 433:717-746. 
Whiting, William S. 

1923. The relation between the clumps of bacteria found in market milk and 

the flora of dairy utensils. New York State (Geneva) Agr. Exp. 

Sta., Tech. Bui. 98:3-36. 
Breed, E. S. 

1926. The microscopic appearance of market milk and cream. New York 

State (Geneva) Agr. Exp. Sta., Tech. Bui. 120:3-7. 
Newman, E. W. 

1927. One solution technique for direct microscopic counting of bacteria in 

milk. Proc. Soc. Exp. Biol, and Med., 24:323-325. 

Reductase Method 
Hastings, E. G. 

1919. The comparative value of quantitative and qualitative bacteriological 

methods as applied to milk with especial consideration of the methy- 
lene blue reduction test. Jour. Dairy Sci., 2:293-311. 



28 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION 

Hastings, E. G., A. Davenport, and W. H. Wright. 

1922. The influence of certain factors on the methylene blue reduction test 
for determining the number of bacteria in milk. Jour. Dairy Sci., 
5:438-454. 

Little-plate Method 
Frost, W. D. 

1916. Comparison of a rapid method of counting bacteria in milk with the 

standard plate method. Jour. Infect. Diseases, 19:273-287. 

1917. Counting the living bacteria in milk: a practical test. Jour. Bact., 

2:567-583. 



STATION PUBLICATIONS AVAILABLE FOR FREE DISTRIBUTION 



No. 

253. Irrigation and Soil Conditions in the 
Sierra Nevada Foothills, California. 

262. Citrus Diseases of Florida and Cuba 

Compared with those of California. 

263. Size Grades for Ripe Olives. 

268. Growing and Grafting Olive Seedlings. 

273. Preliminary Report on Kearney Vine- 
yard Experimental Drain, Fresno 
County, California. 

276. The Pomegranate. 

277. Sudan Grass. 

278. Grain Sorghums. 

279. Irrigation of Rice in California. 
283. The Olive Insects of California. 
294. Bean Culture in California. 

304. A Study of the Effects of Freezes on 

Citrus in California. 
310. Plum Pollination. 

312. Mariout Barley. 

313. Pruning Young Deciduous Fruit 

Trees. 
319. Caprifigs and Caprification. 

324. Storage of Perishable Fruit at Freez- 

ing Temperatures. 

325. Rice Irrigation Measurements and 

Experiments in Sacramento Valley, 

1914-1919. 
328. Prune Growing in California. 
331. Phylloxera-Resistant Stocks. 
335. Cocoanut Meal as a Feed for Dairy 

Cows and Other Livestock. 

339. The Relative Cost of Making Logs 

from Small and Large Timber. 

340. Control of the Pocket Gopher in 

California. 

343. Cheese Pests and Their Control. 

344. Cold Storage as an Aid to the Mar- 

keting of Plums. 

346. Almond Pollination. 

347. The Control of Red Spiders in Decid- 

uous Orchards. 

348. Pruning Young Olive Trees. 

349. A Study of Sidedraft and Tractor 

Hitches. 

350. Agriculture in Cut-over Redwood 

Lands. 

353. Bovine Infectious Abortion. 

354. Results of Rice Experiments in 1922. 

357. A Self-mixing Dusting Machine for 

Applying Dry Insecticides and 
Fungicides. 

358. Black Measles, Water Berries, and 

Related Vine Troubles. 

361. Preliminary Yield Tables for Second 

Growth Redwood. 

362. Dust and the Tractor Engine. 

363. The Pruning of Citrus Trees in Cali- 

fornia. 

364. Fungicidal Dusts for the Control of 

Bunt. 

365. Avocado Culture in California. 

366. Turkish Tobacco Culture, Curing and 

Marketing. 

367. Methods of Harvesting and Irrigation 

in Relation of Mouldy Walnuts. 

368. Bacterial Decomposition of Olives dur- 

ing Pickling. 

369. Comparison of Woods for Butter 

Boxes. 

370. Browning of Yellow Newtown Apples. 

371. The Relative Cost of Yarding Small 

and Large Timber. 

373. Pear Pollination. 

374. A Survey of Orchard Practices in the 

Citrus Industry of Southern Cali- 
formia. 

375. Results of Rice Experiments at Cor- 

tena, 1923. 

376. Sun-Drying and Dehydration of Wal- 

nuts. 

377. The Cold Storage of Pears. 
379. Walnut Culture in California. 



BULLETINS 
No. 



380. 

382. 

385. 
386. 

387. 
388. 

389. 
390. 

391. 

392. 
393. 
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S95. 
396. 

397. 

398. 
399. 



400. 
401. 

402. 
404. 
405. 
406. 
407. 



408. 
409. 



410. 



412. 



414. 



415. 
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427. 
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429. 



Growth of Eucalyptus in California 
Plantations. 

Pumping for Drainage in the San 
Joaquin Valley, California. 

Pollination of the Sweet Cherry. 

Pruning Bearing Deciduous Fruit 
Trees. 

Fig Smut. 

The Principles and Practice of Sun- 
drying Fruit. 

Berseem or Egyptian Clover. 

Harvesting and Packing Grapes in 
California. 

Machines for Coating Seed Wheat with 
Copper Carbonate Dust. 

Fruit Juice Concentrates. 

Crop Sequences at Davis. 

Cereal Hay Production in California. 
Feeding Trials with Cereal Hay. 

Bark Diseases of Citrus Trees. 

The Mat Bean (Phaseolus aconitifo- 
lius). 

Manufacture of Roquefort Type Cheese 
from Goat's Milk. 

Orchard Heating in California. 

The Blackberry Mite, the Cause of 
Redberry Disease of the Himalaya 
Blackberry, and its Control. 

The Utilization of Surplus Plums. 

Cost of Work Horses on California 
Farms. 

The Codling Moth in Walnuts. 

The Dehydration of Prunes. 

Citrus Culture in Central California. 

Stationary Spray Plants in California. 

Yield, Stand and Volume Tables for 
White Fir in the California Pine 
Region. 

Alternaria Rot of Lemons. 

The Digestibility of Certain Fruit By- 
products as Determined for Rumi- 
nants. 

Factors Affecting the Quality of Fresh 
Asparagus after it is Harvested. 

Paradichlorobenzene as a Soil Fumi- 
gant. 

A Study of the Relative Values of Cer- 
tain Root Crops and Salmon Oil as 
Sources of Vitamin A for Poultry. 

Planting and Thinning Distances for 
Deciduous Fruit Trees. 

The Tractor on California Farms. 

Culture of the Oriental Persimmon 
in California. 

Poultry Feeding : Principles and 
Practice. 

A Study of Various Rations for 
Finishing Range Calves as Baby 
Beeves. 

Economic Aspects of the Cantaloupe 
Industry. 

Rice and Rice By-products as Feeds 
for Fattening Swine. 

Beef Cattle Feeding Trials, 1921-24. 

Cost of Producing Almonds in Cali- 
fornia ; a Progress Report. 

Apricots (Series on California Crops 
and Prices). 

The Relation of Rate of Maturity to 
Egg Production. 

Apple Growing in California. 

Apple Pollination Studies in Cali- 
fornia. 

The Value of Orange Pulp for Milk 
Production. 

The Relation of Maturity of Cali- 
fornia Plums to Shipping and 
Dessert Quality. 

Economic Status of the Grape Industry. 



CIRCULARS 

No. No. 

87. Alfalfa. 259. 

117. The Selection and Cost of a Small 261. 

Pumping Plant. 262. 

127. House Fumigation. 263. 

129. The Control of Citrus Insects. 264. 
136. Melilotus indica as a Green-Manure 

Crop for California. 265. 

144. Oidium or Powdery Mildew of the 266. 

Vine. 

157. Control of the Pear Scab. 267. 
164. Small Fruit Culture in California. 

166. The County Farm Bureau. 269. 

170. Fertilizing California Soils for the 270. 

1918 Crop. 272. 
173. The Construction of the Wood-Hoop 

Silo. 273. 

178. The Packing of Apples in California. 276. 

179. Factors of Importance in Producing 277. 

Milk of Low Bacterial Count. 

202. County Organizations for Rural Fire 278. 

Control. 

203. Peat as a Manure Substitute. 279. 
209. The Function of the Farm Bureau. 

212. Salvaging Rain-Damaged Prunes. 281. 
215. Feeding Dairy Cows in California. 
217. Methods for Marketing Vegetables in 

California. 282. 

230. Testing Milk, Cream, and Skim Milk 

for Butterfat. 283. 

231. The Home Vineyard. 284. 

232. Harvesting and Handling California 285. 

Cherries for Eastern Shipment. 286. 

234. Winter Injury to Young Walnut Trees 287. 

during 1921-22. 288. 

238. The Apricot in California. 289. 

239. Harvesting and Handling Apricots 290. 

and Plums for Eastern Shipment. 291. 

240. Harvesting and Handling Pears for 

Eastern Shipment. 292. 

241. Harvesting and Handling Peaches for 293. 

Eastern Shipment. 294. 

243. Marmalade Juice and Jelly Juice from 295. 

Citrus Fruits. 

244 Central Wire Bracing for Fruit Trees. 296. 
245. Vine Pruning Systems. 

248. Some Common Errors in Vine Prun- 298. 

ing and Their Remedies. 

249. Replacing Missing Vines. 300. 

250. Measurement of Irrigation Water on 301. 

the Farm. 302. 

252. Supports for Vines. 303. 

253. Vinevard Plans. 

254. The Use of Artificial Light to Increase 304. 

Winter Egg Production. 305. 

255. Leguminous Plants as Organic Fertil- 306. 

izer in California Agriculture. 

256. The Control of Wild Morning Glory. 307. 

257. The Small-Seeded Horse Bean. 308. 

258. Thinning Deciduous Fruits. 309. 



Pear By-products. 

Sewing Grain Sacks. 

Cabbage Growing in California. 

Tomato Production in California. 

Preliminary Essentials to Bovine 

Tuberculosis Control. 
Plant Disease and Pest Control. 
Analyzing the Citrus Orchard by 

Means of Simple Tree Records. 
The Tendency of Tractors to Rise in 

Front"; Causes and Remedies. 
An Orchard Brush Burner. 
A Farm Septic Tank. 
California Farm Tenancy and Methods 

of Leasing. 
Saving the Gophered Citrus Tree. 
Home Canning. 
Head, Cane, and Cordon Pruning of 

Vines. 
Olive Pickling in Mediterranean Coun- 
tries. 
The Preparation and Refining of Olive 

Oil in Southern Europe. 
The Results of a Survey to Determine 

the Cost of Producing Beef in Cali- 
fornia. 
Prevention of Insect Attack on Stored 

Grain. 
Fertilizing Citrus Trees in California. 
The Almond in California. 
Sweet Potato Production in California. 
Milk Houses for California Dairies. 
Potato Production in California. 
Phylloxera Resistant Vineyards. 
Oak Fungus in Orchard Trees. 
The Tangier Pea. 
Blackhead and Other Causes of Loss 

of Turkeys in California. 
Alkali Soils. 

The Basis of Grape Standardization. 
Propagation of Deciduous Fruits. 
The Growing and Handling of Head 

Lettuce in California. 
Control of the California Ground 

Squirrel. 
The Possibilities and Limitations of 

Cooperative Marketing. 
Coccidiosis of Chickens. 
Buckeye Poisoning of the Honey Bee. 
The Sugar Beet in California. 
A Promising Remedy for Black Measles 

of the Vine. 
Drainage on the Farm. 
Liming the Soil. 
A General Purpose Soil Auger and its 

Use on the Farm. 
American Foulbrood and its Control. 
Cantaloupe Production in California. 
Fruit Tree and Orchard Judging. 



The publications listed above may be had by addressing 

College of Agriculture, 

University of California, 

Berkeley, California. 

10m-ll,'27